Int.J.Curr.Microbiol.App.Sci (2014) 3(5): 77-83
ISSN: 2319-7706 Volume 3 Number 5 (2014) pp. 77-83
http://www.ijcmas.com
Original Research Article
Amelioration of aluminium toxicity on seed germination and early seedling
growth of Pigeon Pea [Cajanus cajan (L.) Millsp.] by 28-Homobrassinolide
M.Madhan, K.Mahesh and S.Seeta Ram Rao*
Department of Botany, Osmania University, Hyderabad 500007, India
*Corresponding author
ABSTRACT
Keywords
Aluminium,
28-homobrassinolide,
Cajanus
cajan,
Germination,
Antioxidative
enzymes
The effect of 28-homobrassinolide on seed germination and seedling growth
of pigeon pea [Cajanus cajan (L.) Millsp.] under aluminium toxicity was
studied. 28-HBL reduced the impact of Al stress on seed germination.
Application of 28-HBL removed the inhibitory influence of Al on seedling
growth. Homobrassinolide application enhanced proline in Al stressed
Cajanus seedlings. Further, the supplementation of 28-HBL to Al stress
treatments increased the activities of antioxidative enzymes viz., catalase
[EC 1.11.1.6]; peroxidase [EC 1.11.1.7]; superoxide dismutase [EC
1.15.1.1] and ascorbate peroxidase [EC 1.11.1.11]. The present studies
demonstrated the ameliorating ability of 28-HBL on the Al induced
inhibition of germination and seedling growth of C.cajan by reducing the
oxidative stress.
Introduction
growth and interfere with water and
mineral nutrient uptake.The use of plant
growth promoting substances potentially
improve the plant growth and productivity
in metal challenged habitats. The
prospects of employing plant growth
regulators in mitigating the metal toxicity
are immense.
Aluminium is the third most abundant
element in earth s crust. Aluminium
toxicity is manifested in acidic conditions
in which phytotoxic form of Al 3+
predominates. Open cast bauxite mining
and
refinement
also
significantly
aggrevating aluminium toxicity. Excess of
aluminium is a major soil constraint to
food and biomass production (Vitorella,
2005). It is estimated that 40% of the
arable soils of the world are acidic and
therefore aluminium poisoining is vary
important
agricultural
problem.
Aluminium toxicity severely impairs root
Brassinosteroids are new group of
phytohormones with significant growth
promoting activity (Rao et al., 2002;
Bajguz, 2007). Brassinosteroids are
regarded as plant growth regulators with
pleiotropic effects as they influence
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Int.J.Curr.Microbiol.App.Sci (2014) 3(5): 77-83
diverse development process such as seed
germination, plant growth, rhizogenesis,
flowering, senescence, and abscission. In
addition, Brassinosteroids also confer
resistance to the plants against abiotic
stresses (Sasse, 2003; Vardhini et al.,
2006). The present study aimed to explore
the possibility of ameliorating aluminium
toxicity in pigeon pea [Cajanus cajan (L)
Millsp] plant by employing 28homobrassinolide a bioactive brassino
steroid.
Twenty seeds from each treatment were
placed in each of 9 cm sterile petri dishes
layered with Whatman No.1 filter paper.
The petri dishes were supplied with 5 ml
of respective test solutions. The seeds
were allowed to germinate in dark at 20
±10C. 3 ml of test solutions were added on
the 4th day of the experiment. Number of
seeds germinated was recorded at the end
of 36, 48 and 60 hours under safe green
light. Emergence of radicle was taken as
the criteria for germination.
Materials and Methods
Growth parameters
On 7th day, seedling growth was recorded
in terms of root and shoot length and their
fresh weight. Root and shoot parts of
seedlings were dried in oven at 1100 C for
24 hours and their dry weights were
recorded.
Chemicals and Seed material
28-Homobrassinolide
(HBL)
was
purchased from CID tech. Research Inc,
Mississauga, Ontario, Canada.The seeds of
pigeon pea [Cajanus cajan (L.) Millsp.]
were procured from National Seed
Corporation, Hyderabad, India.
Free Proline
The amount of proline content was
estimated as described by Bates et al.
(1973). Seedling material (0.5 g) was
homogenized with 10 ml of 3 % (w/v)
sulfosalicylic acid and the homogenate
was filtered through whatman No. 2 filter
paper. The supernatant was taken for
proline estimation. The reaction mixture
was composed of 2 ml of plant extract, 2
ml of acid ninhydrin reagent and 2 ml of
glacial acetic acid. The test tubes
containing above mixture were heated in a
boiling water bath for one hour. The
reaction was terminated in an ice bath
followed by addition of 4 ml of toluene.
The contents were shaken vigorously and
then allowed to separate into phases. The
chromophase containing upper toluene
phase was carefully taken out with the
help of a pipette and the absorbance was
taken at 520 nm. The amount of proline
present was quantified with the help of
proline standard graph.
Aluminum (Al+3) in the form of aluminum
sulphate (Al2 (SO4)3 .16H2O) was used for
the studies. Preliminary experiments were
conducted
employing
different
concentrations of Al and 7.5 mM of Al
was choosen as metal stress concentration,
where seedling growth was found
inhibited considerably but not completely.
Seed Germination and Seedling growth
Seeds of pigeon pea were surface
sterilized with 0.5% (v/v) sodium
hypochlorite from commercially available
(4% NaOCl2) and washed thoroughly with
several changes of sterile distilled water.
They were soaked for 24 hours in either: i)
Distilled water (control) ii) 7.5 mM
Al+3solution (Metal stress control) iii)
0.5µM/1µM/ 2 µM 28-HBL iv) 7.5 mM
Al+3supplemented with 0.5µM/ 1µM / 2
µM 28-HBL.
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Int.J.Curr.Microbiol.App.Sci (2014) 3(5): 77-83
Antioxidant Enzymes
Superoxide dismutase (E.C 1.15.1.1):
Upper part of 7-day-old seedling material
(1 g) was homogenized in 50 mM Tris
HCl (pH 7.5) with addition of 40 mM
phenyl methyl sulfonyl fluoride (PMSF)
and 2 % (w/v) polyvinylpolypyrrolidone
(PVPP). The extract was centrifuged at
15,000g for 20 min and the resultant
supernatant was used for measuring the
following enzyme assays (except for cGCS). The amount of protein in the
enzyme extract was calculated according
to Lowry et al. (1951).
SOD activity was assayed by measuring
its ability to inhibit the photochemical
reduction of nitroblue tetrazolium (NBT)
of Beauchamp and Fridovich (1971).
Three ml of reaction mixture contained 40
mM phosphate buffer (PH=7.8), 13 mM
methionine, 75 µM nitroblue tetrazolium,
0.1 mM EDTA, 0.1 ml of enzyme extract
and 2 µM riboflavin. Riboflavin was
added at the end.
After mixing the contents, test tubes were
shaken and placed 30 cm below light
source consisting of two 15 watt
fluorescent tubes. The reaction was started
by switching on the lights. The reaction
was allowed to take place for 30 minutes
and was stopped by switching off the
lights. A tube with protein kept in the dark
served as blank, while the control tube was
without the enzyme and kept in the light.
The absorbance was measured at 540 nm.
The activity of superoxide dismutase is the
measure of NBT reduction in light without
protein minus NBT reduction in light with
protein. One unit of activity is the amount
of protein required to inhibit 50% initial
reduction of NBT under light.
Catalase (CAT, E.C.1.11.1.6.) activity
was determined following Aebi (1974).
The reaction mixture consisted of 50 mM
phosphate buffer, 0.1mM H2O2 and
enzyme extract. The rate of H2O2
decomposition at 240 nm was measured
spectrophotometrically and calculated
using a molar extinction coefficient of
45.2 mM 1 cm 1. One unit of catalase
activity was assumed as the amount of
enzyme that decomposed 1 µmol of H2O2
per mg of soluble protein per minute at 30
0
C.
Peroxidase (POD, E.C.1.11.1.7) activity
was assayed by employing the procedure
of Kar and Mishra (1976). To 0.5 ml of
enzyme extract, 2.5 ml of 0.1 M phosphate
buffer (pH 7), 1 ml of 0.01 M pyrogallol
and 1 ml of 0.005 M H2O2 were added. A
blank was prepared with 0.5 ml of enzyme
extract, 3.5 ml of 0.1 M phosphate buffer
and 1 ml of 0.005 M H2O2. After 5
minutes of incubation at 25 0C, the
reaction was stopped by adding 1 ml of 2.5
N H2SO4. The amount of purpurogallin
formed was estimated by measuring the
absorbance at 420 nm against a blank. The
enzyme activity was expressed as change
in absorbance Units mg-1 protein min-1.
Ascorbate peroxidase (APX; E.C
1.11.1.11) was assayed by the method of
Nakano and Asada (1981) The reaction
mixture contained 1.5 ml of 50 mM
sodium phosphate buffer (pH 7), 0.2 mM
EDTA, 0.5 ml of 0.5 mM ascorbic acid,
0.5 ml 0.5 mM H2O2 and 0.5 ml of
enzyme sample. The activity was recorded
as the decrease in absorbance at 290 nm
for 1 minute and the amount of ascorbate
oxidized was calculated from the
extinction coefficient of 2.6 mM-1cm-1.
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Int.J.Curr.Microbiol.App.Sci (2014) 3(5): 77-83
Feeding of seedlings with 28-HBL further
enhanced the proline content in Al stressed
seedlings. Though a minor constituent of
the amino acid pool, proline as an
osmolyte act as cellular protector in
several plant species in response to abiotic
stress and scavenge ROS (Ahraf and
Fooland 2007). Such an increase in proline
levels under metal stress due to
brassinosteroids application was also
observed by Choudary et al, (2011) and
Ramakrishna and Rao (2012).
Results and Discussion
Al3+ toxicity inhibited the seed
germination in C.cajan (Table 1).However
application of 28-HBL reduced the toxic
effects of Al3+ on seed germination. The
promotion of seed germination by 28-HBL
was found to be dose dependent. At 2 µM
concentration, HBL not only negated the
toxic influence of Al on seed germination
but
also
further
promoted
seed
germination.
Due to Al toxicity, the activity of
antioxidant enzymes- SOD, CAT, POD
and APX were found increased in pigeon
pea seedlings (Table 3). Application of 28HBL caused further increase in the
activities of the oxidizing enzymes in
seedlings growing under Al toxicity. Al
toxicity cause oxidative damage to the
plant system by activating the production
of reactive oxygen species (Shah et al
2001). These ROS like superoxide radical
(O2-), hydroxyl radicals (OH-), singlet
oxygen (1O2) and hydrogen peroxide
(H2O2) generally are detoxified by
enzymatic antioxidant system. ROS if not
detoxified causes serious damage to macro
molecules such as proteins, lipids and
nucleic acids. In order to scavenge ROS
and to combat oxiadative stress, plants
evolved an efficient antioxidant defense
system.SOD constitutes the first line of
defense against ROS in plants. This
enzyme catalyzes the detoxification of O2to H2O and O2 (Alscher et al.2002). CAT
and POD further breakdown H2O2 to H2O
and O2. Further, Ascorbate peroxidase
(APX) which uses reduced ascorbate as a
reductant in the first step of ascorbateglutathione cycle is recognized as
important
peroxidase
in
H2O2
detoxification. Application of HBL to Al
Aluminium
toxicity
resulted
in
suppression of seedling growth (Table 2).
Al toxicity reduced the growth in terms of
length, fresh and dry weight of root and
shoot (Table 2). The impact of Al toxicity
was much more pronounced on root
growth. However application of 28-HBL
improved the seedling growth in seedlings
challenged with toxic levels of Al. 28HBL at 2µM concentration, caused a
considerable increase in seedling growth
under Al stress and restored the growth to
the level of unstressed control seedlings.
The growth promoting effects of BRs on
seedlings under stress conditions might be
attributed to their involvement in cell
elongation and cell cycle progression
(Gonzalez-Gracia et al.2011) as well as
regulation of genes encoding xyloglucan
endotransglucosylase/hydrolase (XTHs),
expansions, glucanases, sucrose synthase
and cellulose synthase or by activating the
H-ATPase
activity
(Ashraf
et
al.2010).Similarly exogenous application
of BRs improved the growth of Nistressed maize seedlings (Bhardwaj et
al.2007) and Cr- stressed radish seedlings
(Choudary et al.2011) and Zn stressed
radish seedlings (Ramakrishna and Rao,
2013).
The levels of proline increased in the
seedlings under Al stress (Table 3).
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Int.J.Curr.Microbiol.App.Sci (2014) 3(5): 77-83
Table.1 Effect of 28-homobrassinolde on germination of Cajanus cajan seeds subjected to
Aluminium toxicity (Results expressed as % seed germination)
Treatment
Control
0.5 M HBL
1 M HBL
2 M HBL
Al (7.5 mM)
Al+0.5 M HBL
Al+1 M HBL
Al+2 M HBL
36 Hours
15
14
17
20
9
17
19
20
48 Hours 60 Hours
43
85
48
87
51
89
61
86
31
80
37
86
52
88
58
91
The data presented above are Mean ± S.E. (n=5). Al+3: 7.5mM Aluminium; HBL= 28homobrassinolide
Table.2 Effect of 28-homobrassinolide alone treatments and in combination with
Al+3 stress on Cajanus seedling growth
Treatment
Control
0.5µM HBL
1.0 µM HBL
2.0 µM HBL
Al3+(7.5 mM)
Al3++0.5µM HBL
Al3++1.0 µM HBL
Al3++2.0 µM HBL
Root
length
(cm)
Shoot
length
(cm)
Root FW
(mg)
Root DW
(mg)
Shoot FW
(mg)
Shoot DW
(mg)
5.12±0.70
6.28± 0.24
8.10± 0.26
9.30± 1.09
0.50±0.24
2.87±0.87
4.23±0.12
5.28± 0.34
5.50±0.51
6.51±0.54
7.91±0.89
8.88± 0.45
3.11±0.59
4.20±0.91
4.82±0.29
5.91±0.78
884.22±3.98
1141.34±9.2
1408.12±11.6
1601.23±23.1
87.77±7.54
504.11±0.86
755.32±0.92
929.23±0.11
267.2±5.05
372.2±10.2
427.9±12.1
486.9±14.3
27.0±6.0
157.9±7.2
232.0±30.1
294.0±12.2
952.3±12.0
1129.4±45.0
1376.1±23.0
1541.3±27.0
487.0±23.0
710.1±11.2
823.3±19.7
994.7±10.0
275.22±12.0
327.50±13.3
428.32±42.0
490.83±12.0
151.25±23.0
210.98±17.1
245.15±25.1
301.72±15.6
The data presented above are Mean ± S.E. (n=5). Al+3: 7.5mM Aluminium; HBL= 28homobrassinolide.
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Int.J.Curr.Microbiol.App.Sci (2014) 3(5): 77-83
Table.3 Effect of 28-homobrassinolide alone treatments and in combination with Al+3 stress
on
content of free proline and antioxidant enzyme activities (CAT: catalase, POD:
peroxidase, SOD: super oxide dismutase and APX: ascorbate peroxidase)
Treatments
Control
0.5µM HBL
1.0 µM HBL
2.0 µM HBL
Al3+(7.5 mM)
Al3++0.5µM HBL
Al3++1.0 µM HBL
Al3++2.0 µM HBL
Free
Proline
(mg g-1FW)
1.25±0.20
2.11±0.27
1.93±0.12
2.07±0.13
2.50±0.16
2.54±0.12
2.98±0.13
3.15±0.07
CAT
(U mg-1 protein
min-1)
0.84±0.70
1.72±0.56
2.65±0.70
2.07±0.13
1.82±0.73
1.82±0.57
2.35±1.06
2.78±1.41
POD
(U mg-1 protein
min-1)
0.028±0.007
0.031±0.008
0.032±0.005
0.038±0.008
0.021±0.004
0.024±0.005
0.027±0.002
0.029±0.007
SOD
(U mg-1
protein min-1)
1.208±0.08
1.689±0.48
1.911±0.65
2.373±0.60
2.516±0.25
3.013±0.44
3.050±0.14
3.428±0.14
APX
(µmol ASA mg-1
protein min-1)
5.39±0.35
5.84±0.17
6.33±0.51
6.72±0.42
7.83±0.48
8.49±0.50
9.28±0.61
9.69±0.52
The data presented above are Mean ± S.E. (n=5). Al+3: 7.5mM Aluminium; HBL= 28homobrassinolide.
References
stressed seedlings enhanced the APX
activity,
indicating
the
efficient
scavenging of H2O2 thereby presenting the
H2O2 mediated cell damage. Similarly,
Bajguz (2010) observed that exogenous
application of brassinosteroid increased
the activity of APX in Chlorella cultures
under heavy metal stress. The study
clearly showed that application of 28-HBL
to stressed seedlings enhanced the
antioxidative defense system in plants.
Aebi, H. 1974). Catalase. In: Bergmeyer,
HU ed.), Methods of Enzymatic
Analysis. Verlag
Chemie/Academic
Press Inc. Weinheim/NewYork. pp.
673 680.
Alscher, R.G., Erturk, N. and Heath, L.S.
2002. Role of superoxide dismutase s
SODs) in controlling oxidative stress in
plants .J. of Exp Bot, 53:1331-1341.
Ashraf, M. and Foolad, M.R. 2007. Roles of
glycinebetaine and proline in improving
plant abiotic stress resistance. Environ
Exp Bot, 59:206 216.
Ashraf, M., Akram, N.A., Arteca, R.N. and
Foolad, M.R. 2010. The physiological,
biochemical and molecular roles of
brassinosteroids and salicylic acid in
plant processes and salt tolerance. Crit
Rev of Plant Sci, 29:162-190.
Bajguz,
A.
2007.
Metabolism
of
brassinosteroids in Plants. Plant Physiol.
Biochem., 45:95-107
Bajguz, A. 2010. An enhanced effect of
brassinolide on the growth and
antioxidant activity in Chlorella cultures
under heavy metal stress. Environ Exp
Bot. 68:175-179.
The results of the study revealed the
capability of 28-homobrassinolide is
counteracting the toxicity of aluminium on
seed germination and seedling growth.
The alleviation of Al toxicity stress by 28HBL was found associated with furthering
the enzymatic antioxidant defense
mechanism.
Acknowledgement
The financial assistance from University
Grants Commission, New Delhi, India is
gratefully acknowledged.
82
Int.J.Curr.Microbiol.App.Sci (2014) 3(5): 77-83
Bates, L., Waldren, R.P. and Teare, I.D.
1973. Rapid determination of free
proline for water-stress studies. Plant
and Soil, 39:205-207.
Beauchamp, C. and Fridovich, I.
1971.Superoxide dismutase improved
assay and an assay applicable to
acrylamide gels. Anal. Bioche, 44:276287.
Bhardwaj, R., Arora, N., Sharma, P. and
Arora, H.K. 2007. Effect of 28homobrassinolide on seedling growth,
lipid peroxidation and antioxidative
enzyme activities under nickel stress in
seedlings of Zea mays L. Asi. J. of Plant
Sci. 6:765-772.
Choudhary, S.P., Kanwar, M., Bhardwaj, R.,
Gupta, B.D. and Gupta, R.K. 2011.
Epibrassinolide ameliorates Cr VI)
stress via influencing the levels of
indole-3-acetic acid, abscisic acid,
polyamines and antioxidant system of
radish seedlings. Chemosphere, 84:592600.
Gonzalez-Gracia, M.P., Vilarrsa-Blasi, J.,
Zhiponova, M., Divol, F., Mora-Garcia,
S., Russinova, E. and Cano-Delgado,
A.L. 2011. Brassinosteroids control
meristem size by promoting cell cycle
progression in Arabidopsis roots.
Development. 138:849-859.
Kar, M. and Mishra, D. 1976. Catalase
peroxidase and polyphenol oxidase
activities during rice leaf senescence.
Plant Physiol, 57:315-319.
Lowry, O.H., Rosebrough, N.J., Farr, A.L.
and Randall, R.J. 1951. Protein
measurement with the Folin phenol
reagent. J. of Biol. Chem, 193: 265-275.
Michalak, A. 2006. Phenolic compounds
and their antioxidant activity in plants
growing under heavy metal stress.
Polish J Environ Stud. 15:523-530.
Nakano, Y. and Asada, K. 1981. Hydrogen
peroxide is scavenged by ascorbate
specific
peroxidase
in
spinach
chloroplasts. Plant Cell Physiol, 22:
867 880.
Ramakrishna, B. and Rao, S.S.R. 2012. 24Epibrassinolide alleviated zinc-induced
oxidative stress in radish Raphanus
sativus L.) seedlings by enhancing
antioxidative system. Plant Growth
Regul, 68:249-259.
Ramakrishna, B. and Rao, S.S.R. 2013. 24Epibrassinolide maintains elevated
redox state of AsA and GSH in radish
Raphanus sativus L.) seedlings under
zinc stress. Acta Physiol Plant. 35:12911302
Rao, S.S.R., Vardhini, B.V., Sujatha, E. and
Anuradha, S. 2002. BrassinosteroidsNew class of phytohormones. Current
Sci., 82:1239-1245.
Sasse, J.M. 2003. Physiological actions of
brassinosteroids: an update. J Plant
Growth Regul.22:276-288.
Shah, K., Kumar, R.G., Verma, S. and
Dubey, R.S. 2001. Effect of cadmium on
lipid peroxidation, superoxide anion
generation and activities of antioxidant
enzymes in growing rice seedling. Plant
Sci, 161:1135 1144
Vardhini, B.V., anuradha, S. and Rao, S.S.R.
2006. Brassinosteroids-new class of
plant hormone with potential to improve
crop productivity. Ind. J. of Plant
Physiol., 11: 1-12.
Vitorello, V.A., Capaldi, F.R.C. and
Stefanuto, V.A. 2005).Recent advances
in aluminum toxicity and resistance in
higher plants. Braz. J. of Plant Physiol,
17: 129-143.
83